Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available here click here .

Problem with the multi-channel pipette during the addition of the conjugate or substrate:

Check pipette calibration and for bubbles during pipetting. Practice pipetting technique. A video providing tips on proper pipetting technique is available here .

Poor standard curve, does not match certificate of analysis

Inadequate plate washing:

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here.

Poor pipetting technique:

Check pipette calibration and for bubbles during pipetting. Practice pipetting technique. A video providing tips on proper pipetting technique is also available in the Learning Center section of the website or click here.

Inadequate mixing of reagents:

Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc) to ensure reagents are homogenous before use. Confirm the correct volumes were used if mixing kit reagents to make a “working” component for the assay.

Inadequate shaking of plate:

Incorrect shaking motion can affect proper mixing and antibody/analyte interaction. Due to various equipment models please refer to the Learning Center section of the website for a diagram indicating the appropriate shaking method or click-here.

Wrong volume of reagents added to the well:

Check residual volumes to confirm correct volume of reagents were added to the wells.

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here

Contamination of the substrate with enzyme conjugate:

Ensure all containers are clean and clearly marked. Check TMB for blue coloring. If TMB has turned blue, do not use in your assay and contact product support. Also do not expose substrate to light before use.

High background OD readings (Blank or 0 STD values are too high)

Automated plate washer is not functioning properly and is not washing all wells in the same manner:

Ensure that wells are all equally being washed in the same manner with appropriate wash volume, refrain from using multi-channel pipette for washing instead use a more vigorous method of washing. A video displaying proper washing technique is also available in the Learning Center section of the website or click here

Omission of a wash step

Old or contaminated wash buffer:

Make fresh wash buffer.

Contamination of the substrate with enzyme conjugate:

Ensure all containers are clean and clearly marked. Check TMB for blue coloring. If TMB has turned blue, do not use in your assay and contact product support. Also do not expose substrate to light before use.

Wrong conjugate dilution:

A higher than normal concentration may result in an elevated background. Check residual volumes and confirm conjugate was prepared correctly.

Wrong filter used in the plate reader:

Confirm the correct filter indicated in the protocol is being used when reading the plate.

Follow method in protocol for appropriate sample prep or call ALPCO tech support for clarification.

Omission of a reagent:

Check residual volumes to confirm correct volume of each reagent was added to the wells.

Dilution error:

Double check dilution calculations and the volumes used in making these dilutions. If needed, remake the dilutions.

Sample IDs mixed up:

Review sample ID’s to confirm they were labeled correctly.

Wrong curve fit used:

Use the curve fit recommended in the protocol. If more than one recommendation is provided use the regression method that best fits the standard data points. Also, it is very difficult to generate a 4 or 5 parameter curve with Excel and the use of a software program for this type of regression is recommended.

No color development or very low OD readings

Wrong filter used in the plate reader:

Confirm the correct filter indicated in the protocol is being used when reading the plate.

Inadequate mixing of reagents:

Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc) to ensure reagents are homogenous before use. Confirm the correct volumes were used if mixing kit reagents to make a “working” component for the assay.

Inadequate shaking of plate:

Incorrect shaking motion can affect proper mixing and antibody/analyte interaction. Due to various equipment models please refer to the Learning Center section of the website for a diagram indicating the appropriate shaking method or click-here.

Reagents contaminated or added in the wrong order:

Ensure all containers are clean and clearly marked and follow the steps as instructed in the protocol.

Assay incubation times not followed correctly:

Follow the incubation times and temperatures as indicated in the protocol. Modifying either factor will affect the assay kinetics and may lead to erroneous results.

Improperly prepared sample:

Follow method in protocol for appropriate sample prep or call ALPCO tech support for clarification.

Verify the appropriate standard curve was used and check results against the values given in the certificate of analysis.

Incorrect reconstitution volume used:

Follow protocol as instructed. Allow reconstituted reagents to sit for the length of time indicated in the manual before use. Use appropriate equipment (vortex mixer, etc.) to ensure reagents are homogenous before use.

Wrong curve fit used:

Use the curve fit recommended in the protocol. If more than one recommendation is provided use the regression method that best fits the standard data points. Also, it is very difficult to generate a 4 or 5 parameter curve with Excel and the use of a software program for this type of regression is recommended.

Flow Cytometry

Reducing non-specific binding

Non-specific binding can be due to several reasons.

a) Too much antibody can increase the amount of non-specific binding of your negative population reducing your signal:noise. If the antibody you are using has not been titered, then a titration of your antibody should be done to determine the optimal concentration.

b) Non-specific binding can also be due to Fc-mediated binding. You can use IgG (Lampire) of the same species as your antibody to block non-specific binding. Using monoclonal antibodies specific for Fc Receptors to block Fc-mediated binding can also help reduce background binding.

c) The use of directly conjugated antibodies can also reduce the amount of non-specific binding.

d) The use of a biotinylated antibody with a streptavidin fluorochrome conjugate can be a source of non-specific binding in some cells. Biotin is a component of normal cellular metabolism, and as such, this may be detected in high levels in cells.

e) Dead cells are notorious for non-specifically binding antibodies. Inclusion of a viability dye (i.e. PI, 7-AAD) in your assay will allow you to exclude the dead cell population from your analysis. You must make sure that your viability dye is compatible with the other fluorochromes in your sample. Also, cells cannot be fixed when using a viability dye as this will make them permeable to the dye and all the cells will appear dead.

Infinicyt™: Flow Cytometry Software

Infinicyt™ is a flow cytometry analysis software program that provides an innovative approach for data integration and multidimensional analysis of flow cytometry data. Its state-of-the-art features make analysis and interpretation of results easier, faster and more accurate.

There may be contamination at the head of the column. Change direction of the column and rinse for 30 minutes at a low flow rate (0.2ml/min) with mobile phase.

There may be air in the system. Degas pump.

Vials may be contaminated. Use new vials or clean with methanol.

Variable retention times

There may be a drift in temperature. Use a column oven.

Imprecise pump delivery. Check pumps and degas system if needed.

System is not in steady state yet. Rinse system mobile phase for 15 minutes.

Baseline is drifting

Detector lamp may not have reached working temperature. Wait until appropriate temperature is reached.

Detector lamp may be too old and needs to be replaced.

System is not in steady state yet. Rinse system mobile phase for 15 minutes.

Imprecise pump delivery. Check pumps and degas system if needed.

Baseline is not smooth

Imprecise pump delivery. Check pumps and degas system if needed.

Detector flow cell is dirty. Try cleaning flow cell.

Chemiluminescence

Chemiluminescent Plate Reader Settings

Reader Settings

Please contact the microplate reader manufacturer’s technical services department for additional assistance. The instrument settings below are meant to serve as a guideline. It is optional to shake the plates before reading for less than or equal to 3 seconds.